Tissue repair fabric

ABSTRACT

This invention is directed to prosthesis, which, when implanted into a mammalian patient, serves as a functioning replacement for a body part, or tissue structure, and will undergo controlled biodegradation occurring concomitantly with bioremodeling by the patient&#39;s living cells. The prosthesis is treated so that it is rendered non-antigenic so as not to elicit a significant humoral immune response. The prosthesis of this invention, in its various embodiments, thus has dual properties. First, it functions as a substitute body part, and second, it functions as bioremodeling template for the ingrowth of host cells.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention is in the field of implantable biologicalprostheses. The present invention is a non-antigenic, resilient,completely bioremodelable, biocompatible tissue prosthesis which can beengineered into a variety of shapes and used to repair, augment, orreplace mammalian tissues and organs. Each layer of the prosthesis isgradually degraded and remodeled by the host's cells which replace theimplanted prosthesis in its entirety to restore structure and functionand is useful for organ repair and reconstruction. Thus, the prosthesisacts as a template by which the host's cells will remodel themselvesthrough a process that will replace the prosthetic collagen moleculeswith the appropriate host cells in order to restore and replace theoriginal host tissue or organ.

[0003] 2. Brief Description of the Background of the Invention

[0004] Despite the growing sophistication of medical technology,repairing and replacing damaged tissues remains a frequent, costly, andserious problem in health care. Currently implantable prostheses aremade from a number of synthetic and treated natural materials. The idealprosthetic material should be chemically inert, non-carcinogenic,capable of resisting mechanical stress, capable of being fabricated inthe form required, and sterilizable, yet not be physically modified bytissue fluids, excite an inflammatory or foreign body reaction, induce astate of allergy or hypersensitivity, or, in some cases, promotevisceral adhesions (Jenkins S. D., et al. Surgery 94(2):392-398, 1983).

[0005] For example, body wall defects that cannot be closed withautogenous tissue due to trauma, necrosis or other causes requirerepair, augmentation, or replacement with synthetic mesh. In reinforcingor repairing abdominal wall defects, several prosthetic materials havebeen used, including tantalum gauze, stainless steel mesh, DACRON®,ORLON®, FORTISAN®, nylon, knitted polypropylene (MARLEX®), microporousexpanded-polytetrafluoroethylene (GORE-TEX®), dacron reinforced siliconerubber (SILASTIC®), polyglactin 910 (VICRYL®), polyester (MERSWENE®),polyglycolic acid (DEXON®), processed sheep dermal collagen (PSDC®),crosslinked bovine pericardium (PERI-GUARD®), and preserved human dura(LYODURA®). No single prosthetic material has gained universalacceptance.

[0006] The major advantages of metallic meshes are that they are inert,resistant to infection and can stimulate fibroplasia. Their majordisadvantage is the fragmentation that occurs after the first year ofimplantation as well as the lack of malleability. Synthetic meshes havethe advantage of being easily molded and, except for nylon, retain theirtensile strength in the body. European Patent No. 91122196.8 to Krajicekdetails a triple-layer vascular prosthesis which utilizesnon-resorbable, synthetic mesh as the center layer. The synthetictextile mesh layer is used as a central frame to which layers ofcollagenous fibers can be added, resulting in the tri-layered prostheticdevice. The major disadvantage of a non-resorbable synthetic mesh islack of inertness, susceptibility to infection, and interference withwound healing.

[0007] In contrast to the non-resorbable mesh disclosed in Krajicek(E.P. No. 91122196.8), absorbable synthetic meshes have the advantage ofimpermanence at the site of implantation, but often have thedisadvantage of losing their mechanical strength, because of dissolutionby the host, prior to adequate cell and tissue ingrowth.

[0008] The most widely used material for abdominal wall replacement andfor reinforcement during hernia repairs is MARLEX®; however, severalinvestigators reported that with scar contracture, polypropylene meshgrafts became distorted and separated from surrounding normal tissue ina whorl of fibrous tissue. Others have reported moderate to severeadhesions when using MARLEX®.

[0009] GORE-TEX® is currently believed to be the most chemically inertpolymer and has been found to cause minimal foreign body reaction whenimplanted. A major problem exists with the use ofpolytetrafluoroethylene in a contaminated wound as it does not allow forany macromolecular drainage, which limits treatment of infections.

[0010] Collagen first gained utility as a material for medical usebecause it was a natural biological prosthetic substitute that was inabundant supply from various animal sources. The design objectives forthe original collagen prosthetics were the same as for synthetic polymerprostheses; the prosthesis should persist and essentially act as aninert-material. With these objectives in mind, purification andcrosslinking methods were developed to enhance mechanical strength anddecrease the degradation rate of the collagen (Chvapil, M., et al (1977)J. Biomed. Mater. Res. 11: 297-314; Kligman, A. M., et al (1986) J.Dermatol. Surg. Oncol. 12 (4): 351-357; Roe, S. C., et al. (1990).Artif. Organs. 14: 443-448. Woodroff, E. A. (1978). J. Bioeng. 2: 1-10).Crosslinking agents originally used include glutaraldehyde,formaldehyde, polyepoxides, diisocyanates (Borick P. M., et al. (1964)J. Pharm. Sci. 52: 1273-1275), and acyl azides. Processed dermal sheepcollagen has been studied as an implant for a variety of applications.Before implantation, the sheep dermal collagen is typically tanned withhexamethylenediisocyanate (van Wachem, P. B., et al. Biomaterials12(March):215-223, 1991) or glutaraldehyde (Rudolphy, V. J., et al. AnnThorac Surg 52:821 -825, 1991). Glutaraldehyde, probably the most widelyused and studied crosslinking agent, was also used as a sterilizingagent. In general, these crosslinking agents generated collagenousmaterial which resembled a synthetic material more than a naturalbiological tissue, both mechanically and biologically.

[0011] Crosslinking native collagen reduces the antigenicity of thematerial (Chvapil, M. (1980) Reconstituted collagen. pp. 313-324. In:Viidik, A., Vuust, J. (eds), Biology of Collagen. Academic Press,London; Harjula, A., et al. (1980) Ann. Chir. Gynaecol. 69: 256-262.) bylinking the antigenic epitopes rendering them either inaccessible tophagocytosis or unrecognizable by the immune system. There are manyknown methods of crosslinking collagenous materials. U.S. Pat. No.5,571,216 details several methods of achieving crosslinking through theheating and joining of free ends of collagen tendrils. U.S. Pat. No.5,263,983 to Yoshizato details crosslinking by treating collagenouscomposites with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride. Glutaraldehyde is also employed as a reagent incrosslinking (See U.S. Pat. No. 4,787,900 to Yannas; U.S. Pat. No.4,597,762 to Walter). However, data from studies using glutaraldehyde asthe crosslinking agent are hard to interpret since glutaraldehydetreatment is also known to leave behind cytotoxic residues (Chvapil, M.(1980), supra; Cooke, A., et al. (1983) Br. J. Exp. Path. 64: 172-176;Speer, D. P., et al. (1980) J. Biomed. Mater. Res. 14: 753-764; Wiebe,D., et al. (1988) Surgery. 104: 26-33). It is, therefore, possible thatthe reduced antigenicity associated with glutaraldehyde crosslinking isdue to non-specific cytotoxicity rather than a specific effect onantigenic determinants. Glutaraldehyde treatment is an acceptable way toincrease durability and reduce antigenicity of collagenous materials ascompared to those that are noncrosslinked. However, glutaraldehydecrosslinking collagen materials significantly limits the body's abilityto remodel the prosthesis (Roe, S. C., et al. (1990), supra).

[0012] All of the above problems associated with traditional materialsstem, in part, from the inability of the body to recognize any implantas “inert”. Although biologic in origin, extensive chemical modificationof collagen tends to render it as “foreign”. To improve the long termperformance of implanted collagenous devices, it is important to retainmany of the properties of the natural collagenous tissue. In this“tissue engineering”0 approach, the prosthesis is designed not as apermanent implant but as a scaffold or template for regeneration orremodeling. Tissue engineering design principles incorporate arequirement for isomorphous tissue replacement, wherein thebiodegradation of the implant matrix occurs at about the same functionalrate of tissue replacement (Yannas, I. V. (1995) Regeneration Templates.pp. 1619-1635. In: Bronzino, J. D. (ed.), The Biomedical EngineeringHandbook, CRC Press, Inc., Boca Raton, Fla.).

[0013] When such a prosthesis is implanted, it should immediately serveits requisite mechanical and/or biological function as a body part. Theprosthesis should also support appropriate host cellularization byingrowth of mesenchymal cells, and in time, through isomorphous tissuereplacement, be replaced with host tissue, wherein the host tissue is afunctional analog of the original tissue. In order to do this, theimplant must not elicit a significant humoral immune response or beeither cytotoxic or pyrogenic to promote healing and development of theneo-tissue.

[0014] Prostheses or prosthetic material derived from isolated collagenmolecules, either in powder form or in a solution, have beeninvestigated for surgical repair or for tissue and organ replacement.The source of collagen used in these prosthetic devices is determinateof the prostheses' form and function. U.S. Pat. No. 4,787,900 to Yannasdetails a process for the creation of prosthetic blood vessels out of acollagenous composite formed, ex vivo, from individual collagenmolecules in either powder or solution form. The collagenous compound isa conglomerate of individual collagen molecules and does not retain anyof the structural characteristics of the tissue from which the collagenwas originally derived. Instead, this collagenous composite is a“tangled mass of collagen fibrils” that is later chemically tailoredinto the desired shape and thickness required for repairing the specificblood vessel.

[0015] Prostheses or prosthetic material derived from explantedmammalian tissue have been widely investigated for surgical repair orfor tissue and organ replacement. The tissue is typically processed toremove cellular components leaving a natural tissue matrix. Furtherprocessing, such as crosslinking, disinfecting or forming into shapeshave also been investigated. U.S. Pat. No. 3,562,820 to Braun disclosestubular, sheet and strip forms of prostheses formed from submucosaadhered together by use of a binder paste such as a collagen fiber pasteor by use of an acid or alkaline medium. U.S. Pat. No. 4,502,159 toWoodroof provides a tubular prosthesis formed from pericardial tissue inwhich the tissue is cleaned of fat, fibers and extraneous debris andthen placed in phosphate buffered saline. The pericardial tissue is thenplaced on a mandrel and the seam is then closed by suture and the tissueis then crosslinked. U.S. Pat. No. 4,703,108 to Silver provides abiodegradable matrix from soluble collagen solutions or insolublecollagen dispersions which are freeze dried and then crosslinked to forma porous collagen matrix. U.S. Pat. No. 4,776,853 to Klement provides aprocess for preparing biological material for implant that includesextracting cells using a hypertonic solution at an alkaline pH followedby a high salt solution containing detergent; subjecting the tissue toprotease free enzyme solution and then an anionic detergent solution.U.S. Pat. No. 4,801,299 to Brendel discloses a method of processing bodyderived whole structures for implantation by treating the body derivedtissue with detergents to remove cellular structures, nucleic acids, andlipids, to leave an extracellular matrix which is then sterilized beforeimplantation. U.S. Pat. No. 4,902,508 to Badylak discloses a three layertissue graft composition derived from small intestine comprising tunicasubmucosa, the muscularis mucosa, and stratum compactum of the tunicamucosa. The method of obtaining tissue graft composition comprisesabrading the intestinal tissue followed by treatment with an antibioticsolution. U.S. Pat. No. 5,336,616 to Livesey discloses a method ofprocessing biological tissues by treatment of tissue to remove cells,treatment with a cryoprotectant solution, freezing, rehydration, andfinally, innoculation with cells to repopulate the tissue. U.S. Pat. No.4,597,762 to Walter discloses a method of preparing collagenousprostheses through proteolysis, crosslinking with glutaraldehyde,welding and subsequent sterilization of animal hide or other mammaliantissues.

[0016] It is a continuing goal of researchers to develop implantableprostheses which can successfuily be used to replace or to facilitatethe repair of mammalian tissues, such as abdominal wall defects andvasculature, so that the intrinsic strength, resillience, andbiocompatability of the host's own cells may be optimally exploited inthe repair process.

SUMMARY OF THE INVENTION

[0017] The present invention overcomes the difficulties of the materialscurrently available and provides a prosthetic device for use in therepair, augmentation, or replacement of damaged tissues and organs. Thisinvention is directed to a prosthetic material, which, when implantedinto a mammalian host, undergoes controlled biodegradation accompaniedby adequate living cell replacement, or neo-tissue formation, such thatthe original implanted prosthesis is ultimately remodeled and completelyreplaced by host derived tissue and cells. The prosthesis of thisinvention, a material for tissue repair, comprises a non-antigenic,sterile, completely bioremodelable collagenous material derived frommammalian tissue. The prosthesis of this invention utilizespre-existing, naturally-formed layers of biological collagen forsurgical repair or for tissue and organ replacement. Unlike the tissuerepair fabrics that are currently available, which use collagenouscomposites formed from reconstituted individual collagen molecules, thecollagenous tissue of the present invention retains the structuralcharacteristics of the tissue from which it has been derrived. Thiscollagenous tissue of the present invention is able to be layered andbonded together to form multilayer sheets, tubes, or complex shapedprostheses. The bonded collagen layers of the invention are structurallystable, pliable, semi-permeable, and suturable.

[0018] Each layer of the prosthetic material of this invention arecompletely bioremodelable and is replaced by host cells to effectivelybecome a host tissue. Moreover, because the present invention iscomprised of naturally-formed pre-existing collagen layers which havebeen harvested from other mammillian tissues, the risk of a significanthumoral response has been greatly decreased. It is, therefore, an objectof this invention to provide a tissue repair fabric that does notexhibit the above-mentioned shortcomings associated with many of thegrafts now being used clinically.

[0019] Another object is the provision of a prosthetic material thatwill allow for and facilitate tissue ingrowth and/or organ regenerationat the site of implantation that is a sterile, non-pyrogenic, andnon-antigenic material derived from mammalian collagenous tissue.Prostheses prepared from this material, when engrafted to a recipienthost or patient, do not elicit a significant humoral immune response.Instead, the prostheses is accepted into the recipient host or patientas non-foreign material and the bioremodeling can proceed withoutinterference from potential immune responses to foreign materials.Prostheses formed from the material concomitantly undergoes controlledbioremodeling occurring with adequate living cell replacement such thatthe original implanted prosthesis is completely remodeled by thepatient's living cells to form a regenerated organ or tissue.

[0020] A further object of the current invention is to provide a simple,repeatable method for manufacturing a tissue repair fabric.

[0021] Still another object of this invention is to provide a method foruse of a novel multipurpose tissue repair fabric in autografting,allografting, and heterografting indications.

[0022] Still a further object is to provide a novel tissue repair fabricthat can be implanted using conventional surgical techniques.

DETAILED DESCRIPTION OF THE INVENTION

[0023] This invention is directed to a tissue engineered prostheses,which, when implanted into a mammalian host, can serve as a functioningrepair, augmentation, or replacement body part, or tissue structure, andwill undergo controlled biodegradation occurring concomitantly withremodeling by the host's cells. The prosthesis of this invention, in itsvarious embodiments, thus has dual properties: First, it functions as asubstitute body part, and second, while still functioning as asubstitute body part, it functions as a remodeling template for theingrowth of host cells. In order to this, the prosthetic material ofthis invention, a tissue repair fabric, was developed comprisingmammalian derived collagenous tissue that is rendered non-antigenic andis able to be bonded to itself or another. Although the prostheses willbe illustrated through construction of various devices and constructs,the invention is not so limited. It will be appreciated that the devicedesign in its shape and thickness is to be selected depending on theultimate indication for the construct.

[0024] In the preferred embodiment, the collagenous material from whichto form prostheses, or the prosthesis itself, is rendered sterile,non-pyrogenic, and non-antigenic. The prosthesis, when engrafted to arecipient host or patient, does not elicit a significant humoral immuneresponse. An acceptable level of response is one that demonstrates nosignificant increase in antibody titer to collagenous tissue proteinsfrom baseline titer levels when blood serum obtained from a recipient ofa prosthesis is tested for antibodies to proteins in extracts of thecollagenous tissue.

[0025] In the preferred method, the tissue repair material or theprosthesis itself is rendered non-antigenic, while maintaining theability for the prosthesis to concomitantly undergo controlledbioremodeling occurring with adequate living cell replacement. Themethod of preparing a non-antigenic prosthetic collagen material,comprises disinfection of the material by a method to prevent microbialdegradation of the material, preferably by use of a solution comprisingperacetic acid; and crosslinking the disinfected collagen material witha crosslinking agent, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC).

[0026] Also in the preferred embodiment, collagenous tissues derivedfrom the mammalian body are used to make said collagen material.Collagenous tissue sources include, but are not limited to intestine,fascia lata, pericardium, and dura mater. The most preferred materialfor use is the tunica submucosa layer of the small intestine. The tunicasubmucosa is preferably separated, or delaminated, from the other layersof the small intestine. This layer is referred to hereinafter as theIntestinal Collagen Layer (“ICL”). Further, the collagen layers of theprosthetic device may be from the same collagen material, such as two ormore layers of ICL, or from different collagen materials, such as one ormore layers of ICL and one or more layers of facia lata.

[0027] The submucosa, or the intestinal collagen layer (ICL), from amammalian source, typically pigs, cows, or sheep, is mechanicallycleaned by squeezing the raw material between opposing rollers to removethe muscular layers (tunica muscularis) and the mucosa (tunica mucosa).The tunica submucosa of the small intestine is harder and stiffer thanthe surrounding tissue, and the rollers squeeze the softer componentsfrom the submucosa. In the examples that follow, the ICL wasmechanically harvested from porcine small intestine using a Bitterlinggut cleaning machine.

[0028] As the mechanically cleaned submucosa may have some hidden,visibly nonapparent debris that affects the consistency of themechanical properties, the submucosa may be chemically cleaned to removedebris and other substances, other than collagen, for example, bysoaking in buffer solutions at 4° C., or by soaking with NaOH ortrypsin, or other known cleaning techniques. Alternative means employingdetergents such as TRITON X-100™ (Rohm and Haas) or sodiumdodecylsulfate (SDS); enzymes such as dispase, trypsin, or thermolysin;and/or chelating agents such as ethylenediaminetetracetic acid (EDTA) orethylenebis(oxyethylenitrilo)tetracetic acid (EGTA) may also be includedin the chemical cleaning method.

[0029] After cleaning, the (ICL) should be decontaminated ordisinfected, preferably with the use of dilute peracetic acid solutionsas described in U.S. Pat. No. 5,460,962, incorporated herein byreference. Decontamination or disinfection of the material is done toprevent degradation of the collagenous matrix by bacteria or proteolyticenzymes. Other disinfectant solutions and systems for use with collagenare known in the art and can be used so long as after the disinfectiontreatment there is no interference in the ability of the material to beremodeled.

[0030] In a preferred embodiment, the prosthetic device of thisinvention has two or more superimposed collagen layers that are bondedtogether. As used herein, “bonded collagen layers” means composed of twoor more layers of the same or different collagen material treated in amanner such that the layers are superimposed on each other and aresufficiently held together by self-lamination. The bonding of thecollagen layers may be accomplished in a number of different ways: byheat welding or bonding, adhesives, chemical linking, or sutures.

[0031] In the preferred method, and in the examples that follow, the ICLis disinfected with a peracetic acid solution at a concentration betweenabout 0.01 and 0.3% v/v in water, preferably about 0.1%, at aneutralized pH between about pH 6 and pH 8 and stored until use at about4° C. in phosphate buffered saline (PBS). The ICL is cut longitudinallyand flattened onto a solid, flat plate. One or more successive layersare then superimposed onto one another. A second solid flat plate isplaced on top of the layers and the two plates are clamped tightlytogether. The complete apparatus, clamped plates and collagen layers,are then heated for a time and under conditions sufficient to effect thebonding of the collagen layers together. The amount of heat appliedshould be sufficiently high to allow the collagen to bond, but not sohigh as to cause the collagen to irreversibly denature. The time of theheating and bonding will depend upon the type of collagen material layerused, the moisture content and thickness of the material, and theapplied heat. A typical range of heat is from about 50° C. to about 75°C., more typically 60° C. to 65° C. and most typically 62° C. A typicalrange of times will be from about 7 minutes to about 24 hours, typicallyabout one hour. The degree of heat and the amount of time that the heatis applied can be readily ascertained through routine experimentation byvarying the heat and time parameters. The bonding step may beaccomplished in a conventional oven, although other apparatus or heatapplications may be used including, but not limited to, a water bath,laser energy, or electrical heat conduction. Immediately following theheating and bonding, the apparatus is cooled, in air or a water bath, ata range between room temperature at 20° C. and 1° C. Rapid cooling,termed quenching, will immediately, or almost immediately, stop theheating action. To accomplish this step, the apparatus may be cooled,typically in a water bath, with a temperature preferably between about1° C. to about 10° C., most preferably about 4° C. Although coolingtemperatures below 1° C. may be used, care will need to be taken not tofreeze the collagen layers, which may cause structural damage. Inaddition, temperatures above 10° C. may be used in quenching, but if thetemperature of the quench is too high, then the heating may not bestopped in time to sufficiently fix the collagen layers in their currentconfiguration.

[0032] The prosthetic material or multi-layered construct is preferablythen crosslinked. Crosslinking the bonded prosthetic device providesstrength and some durability to the device to improve handlingproperties. Crosslinking agents should be selected so as to produce abiocompatible material capable of being remodeled by host cells. Varioustypes of crosslinking agents are known in the art and can be used suchas ribose and other sugars, oxidative agents and dehydrothermal (DHT)methods. A preferred crosslinking agent is1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Thecrosslinking solution containing EDC and water may also contain acetone.In a preferred embodiment, sulfo-N-hydroxysuccinimide is added to thecrosslinking agent (Staros, J. V., Biochem. 21, 3950-3955, 1982).

[0033] In a preferred embodiment, a method comprising disinfection withperacetic acid and subsequent crosslinking with EDC of the ICL materialis performed to reduce the antigenicity of the material. Theimmunoreactive proteins present in non-sterilized, non-crosslinked ICLare either reduced or removed, or their epitopes have been modified suchthat they no longer elicit a significant humoral immune response. Graftimplants of this material do, however, show an initial transientinflammatory response as a result of a wound healing response. As usedherein, the term “non-antigenic” means not eliciting a significanthumoral immune response in a host or patient in whom a prosthesis isimplanted. An acceptable level of response is one that demonstrates nosignificant increase in antibody titer to collagenous tissue proteinsfrom baseline titer levels when blood serum obtained from a recipient ofa prosthesis is tested for antibodies to proteins in extracts of thecollagenous tissue. For a patient or host previously non-sensitized tocollagenous tissue proteins, the preferable serum antibody titer is 1:40or less.

[0034] Prostheses of the preferred embodiment are also preferablynon-pyrogenic. A prosthesis that is pyrogenic, when engrafted to arecipient host or patient, will cause a febrile reaction in the patient,thus affecting the ability of the prosthesis to be remodeled. Pyrogensare tested by intravenous injection of a solution containing a sample ofmaterial into three test rabbits. A temperature sensing probe ispositioned in the rectum of the rabbits to monitor temperature changes.If there is a rise in temperature in any rabbit above 0.5° C., then thetest for that sample is continued in five more rabbits. If not more thanthree of the eight rabbits show individual rises in temperature of 0.5°C. or more and the sum of the eight individual maximum temperature risesdoes not exceed 3.3° C., the material under examination meets therequirements for the absence of pyrogens. (Pyrogen Test (151), pp.1718-1719. In: The United States Pharmacopeia (USP) 23 The United StatesPharmacopeial Convention, Inc., Rockville, Md.)

[0035] The tissue repair fabric of this invention, functioning as asubstitute body part, may be flat, tubular, or of complex geometry. Theshape of the tissue repair fabric will be decided by its intended use.Thus, when forming the bonding layers of the prosthesis of thisinvention, the mold or plate can be fashioned to accommodate the desiredshape. The tissue repair fabric can be implanted to repair, augment, orreplace diseased or damaged organs, such as abdominal wall defects,pericardium, hernias, and various other organs and structures including,but not limited to, bone, periosteum, perichondrium, intervertebraldisc, articular cartilage, dermis, epidermis, bowel, ligaments, andtendons. In addition, the tissue repair fabric can be used as a vascularor intra-cardiac patch, or as a replacement heart valve.

[0036] Flat sheets may be used, for example, to support prolapsed orhypermobile organs by using the sheet as a sling for the organs. Thissling can support organs such as bladder or uterus.

[0037] Tubular grafts may be used, for example, to replace crosssections of tubular organs such as vasculature, esophagus, trachea,intestine, and fallopian tubes. These organs have a basic tubular shapewith an outer surface and a lurinal surface.

[0038] In addition, flat sheets and tubular structures can be formedtogether to form a complex structure to replace or augment cardiac orvenous valves.

[0039] In addition to functioning as a substitute body part or support,the second function of the prosthesis is that of a template or scaffoldfor bioremodeling. “Bioremodeling” is used herein to mean the productionof structural collagen, vascularization, and epithelialization by theingrowth of host cells at a functional rate about equal to the rate ofbiodegradation of the implanted prosthesis by host cells and enzymes.The tissue repair fabric retains the characteristics of the originallyimplanted prosthesis while it is remodeled by the host into all, orsubstantially all, host tissue, and as such, is functional as an analogof the tissue it repairs or replaces. Thus, each layer of the prosthesisis completely bioremodelable and subsequently replaced by host cells.

[0040] The mechanical properties include mechanical integrity such thatthe tissue repair fabric resists creep during bioremodeling, andadditionally is pliable and suturable. The term “pliable” means goodhandling properties. The term “suturable” means that the mechanicalproperties of the layer include suture retention which permits needlesand suture materials to pass through the prosthesis material at the timeof suturing of the prosthesis to sections of native tissue, a processknown as anastomosis. During suturing, such prostheses must not tear asa result of the tensile forces applied to them by the suture, nor shouldthey tear when the suture is knotted. Suturability of tissue repairfabric, i.e., the ability of prostheses to resist tearing while beingsutured, is related to the intrinsic mechanical strength of theprosthesis material, the thickness of the graft, the tension applied tothe suture, and the rate at which the knot is pulled closed.

[0041] As used herein, the term “non-creeping” means that thebiomechanical properties of the prosthesis impart durability so that theprosthesis is not stretched, distended, or expanded beyond normal limitsafter implantation. As is described below, total stretch of theimplanted prosthesis of this invention is within acceptable limits. Theprosthesis of this invention acquires a resistance to stretching as afunction of post-implantation cellular bioremodeling by replacement ofstructural collagen by host cells at a faster rate than the loss ofmechanical strength of the implanted materials due from biodegradationand remodeling. The tissue repair fabric of the present invention is“semi-permeable,” even though it has been crosslinked. Semi-permeabilitypermits the ingrowth of host cells for remodeling or for deposition ofthe collagenous layer. The “non-porous” quality of the prosthesisprevents the passage of fluids that are intended to be retained by theimplantation of the prosthesis. Conversely, pores may be formed in theprosthesis if the quality is required for an application of theprosthesis.

[0042] The mechanical integrity of the prosthesis of this invention isalso in its ability to be draped or folded, as well as the ability tocut or trim the prosthesis obtaining a clean edge without delaminatingor fraying the edges of the construct.

[0043] Additionally, in another embodiment of the invention,mechanically sheared or chopped collagen fibers can be included betweenthe collagen layers adding bulk to the construct and providing amechanism for a differential rate of remodeling by host cells. Theproperties of the construct incorporating the fibers may be altered byvariations in the length and diameter of the fibers; variations on theproportion of the fibers used, and fully or partially crosslinkingfibers. The length of the fibers can range from 0.1 cm to 5.0 cm.

[0044] In another embodiment of the invention, collagen threads, such asthose disclosed in U.S. Pat. No. 5,378,469 and incorporated herein byreference, can be incorporated into the multilayered tissue repairfabric for reinforcement or for different functional rates ofremodeling. For example, a helix or “twist”, of a braid of 20 to 200denier collagen thread may be applied to the surface of the tissuerepair fabric. The diameter size of the helix or braid of collagenthread can range from 50 to 500 microns, preferably 100 to 200 microns.Thus, the properties of the tissue repair fabric layer can be varied bythe geometry of the thread used for the reinforcement. The functionalityof the design will be dependent on the geometry of the braid or twist.Additionally, collagen thread constructs such as a felt, a flat knittedor woven fabric, or a three-dimensional knitted, woven or braided fabricmay be incorporated between the layers or on the surface of theconstruct. Some embodiments may also include a collagen gel between thelayers alone or with a drug, growth factor or antibiotic to function asa delivery system. Additionally, a collagen gel could be incorporatedwith a thread or a thread construct between the layers.

[0045] As will be appreciated by those of skill in the art, many of theembodiments incorporating collagen gel, thread or a thread constructwill also affect the physical properties, such as compliance, radialstrength, kink resistance, suture retention, and pliability. Physicalproperties of the thread or the thread construct may also be varied bycrosslinking the threads.

[0046] In some embodiments, additional collagenous layers may be addedto the outer or inner surfaces of the bonded collagen layers to create asmooth flow surface for its ultimate application as described in PCTInternational Publication No. WO 95/22301, the contents of which areincorporated herein by reference. This smooth collagenous layer alsopromotes host cell attachment which facilitates ingrowth andbioremodeling. As described in PCT International Publication No. WO95/22301, this smooth collagenous layer may be made from acid-extractedfibrillar or non-fibrillar collagen, which is predominantly type Icollagen, but may also include other types of collagen. The collagenused may be derived from any number of mammalian sources, typicallybovine, porcine, or ovine skin or tendons. The collagen preferably hasbeen processed by acid extraction to result in a fibril dispersion orgel of high purity. Collagen may be acid-extracted from the collagensource using a weak acid, such as acetic, citric, or formic acid. Onceextracted into solution, the collagen can be salt-precipitated usingNaCl and recovered, using standard techniques such as centrifugation orfiltration. Details of acid extracted collagen from bovine tendon aredescribed, for example, in U.S. Pat. No. 5,106,949, incorporated hereinby reference.

[0047] Collagen dispersions or gels for use in the present invention aregenerally at a concentration of about 1 to 10 mg/mL, preferably, fromabout 2 to 6 mg/mL, and most preferably at about 3 to 5 mg/mL and at pHof about 2 to 4. A preferred solvent for the collagen is dilute aceticacid, e.g., about 0.05 to 0.1%. Other conventional solvents for collagenmay be used as long as such solvents are compatible.

[0048] Once the prosthetic device has been produced, it may be airdried, packaged, and sterilized with gamma irradiation, typically 2.5Mrad, and stored. Terminal sterilization employing chemical solutionssuch as peracetic acid solutions as described in U.S. Pat. No.5,460,962, incorporated herein, may also be used.

[0049] In the examples that .follow; the ICL is cut longitudinally andflattened out onto a glass plate, although any inert non-insulated firmmold may be used. In addition, the mold can be any shape: flat, rounded,or complex. In a rounded or complex mold, the bottom and upper moldpieces will be appropriately constructed so as to form the completedprosthesis into the desired shape. Once so constructed, the prosthesiswill keep its shape. Thus, for example, if the prosthesis is formed intoa rounded shape, it can be used as a heart valve leaflet replacement.

[0050] The multi-layered tissue repair fabric may be tubulated byvarious alternative means or combinations thereof. The multilayeredtissue repair fabric may be formed into a tube in either the normal orthe everted position. The tube may be made mechanically by suturing,using interrupted sutures with suitable suture material and isadvantageous as it allows the tube to be trimmed and shaped by thesurgeon at the time of implantation without unraveling. Other processesto seam the submucosa may include adhesive bonding, such as the use offibrin-based glues or industrial-type adhesives such as polyurethane,vinyl acetate or polyepoxy. Preferably heat bonding techniques may alsobe used including laser welding or heat welding of the seam, followed byquenching, to seal the sides of the thus formed tube. Other mechanicalmeans are possible, such as using pop rivets or staples. With thesetubulation techniques, the ends of the sides may be either butt ended oroverlapped. If the sides are overlapped, the seam may be trimmed oncethe tube is formed. In addition, these tubulation techniques aretypically done on a mandrel so as to determine the desired diameter.

[0051] The thus formed structural tube can be kept on a mandrel or othersuitable spindle for further processing. To control functional rates ofbiodegradation and therefore the rate of prosthesis strength decreaseduring bioremodeling, the prosthesis is preferably crosslinked, using asuitable * crosslinking agent, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiumide hydrochloride (EDC). Crosslinking the prosthesis also aidsin preventing luminal creep, in keeping the tube diameter uniform, andin increasing the burst strength. The bond strength of a seam ormultilayer prosthesis is increased when heat or dehydration bondingmethods are used. It is believed that crosslinking the intestinalcollagen layer also improves the suture retention strength by improvingresistance to crack propagation.

[0052] Collagen may be deposited on the internal or external surface ofthe ICL as described in Example 5 of U.S. Pat. No. 5,256,418,incorporated herein by reference. Briefly, when the tissue repair fabricis to be tubulated, the multi-layered fabric is fitted at one end byluer fittings and the collagen dispersion fills the tube. This step mayalso be accomplished as described in the above-referenced patentapplication using a hydrostatic pressure head. The inner layer ofcollagen can also be deposited by flowing collagen into both ends of thetube simultaneously. The tube is then placed into a bath of 20%polyethylene glycol (PEG) in isotonic phosphate buffered saline (PBS),neutral pH. The osmotic gradient between the internal collagen solutionand outer PEG solution in combination cause a simultaneous concentrationand deposition of the collagen along the lumen of the internalstructural layer wall. The tube is then removed from the PEG bath, and aglass rod with a diameter desired diameter of the prosthesis lumen isinserted into the collagen solution, or alternatively, one end of theprosthesis is closed and air pressure is applied internally to keep thetube lumen open. The prosthesis is then allowed to dry and subsequentlyis rehydrated in PBS. The thus formed collagen coating, in the form of adense fibrillar collagen, fills slight irregularities in the intestinalstructural layer, thus resulting in a prosthesis with both a smooth flowsurface and a uniform thickness. The procedure also facilitates thebonding of the collagen gel to the intestinal collagen layer. Acollagenous layer of varying thickness and density can be produced bychanging the deposition conditions which can be determined by routineparameter changes. The same procedures can be used to apply the collagento the outer surface of the ICL to create a three-layer prosthesis.

[0053] The prosthesis construct is thrombogenic in small diameter bloodvessel replacements. It can only be used in vascular applications inhigh flow (large diameter) vessels. Therefore, the prosthesis must berendered non-thrombogenic if to be useful for small diameter bloodvessel repair or replacement.

[0054] Heparin can be applied to the prosthesis, by a variety ofwell-known techniques. For illustration, heparin can be applied to theprosthesis in the following three ways. First, benzalkonium heparin(BA-Hep) solution can be applied to the prosthesis by dipping theprosthesis in the solution and then air-drying it. This procedure treatsthe collagen with an ionically bound BA-Hep complex. Second, EDC can beused to activate the heparin, then to covalently bond the heparin to thecollagen fiber. Third, EDC can be used to activate the collagen, thencovalently bond protamine to the collagen and then ionically bondheparin to the protamine. Many other coating, bonding, and attachmentprocedures are well known in the art which could also be used.

[0055] Treatment of the tissue repair fabric with drugs in addition toor in substitution for heparin may be accomplished. The drugs mayinclude for example, growth factors to promote vascularization andepithelialization, such as macrophage derived growth factor (MDGF),platelet derived growth factor (PDGF), endothelial cell derived growthfactor (ECDGF); antibiotics to fight any potential infection from thesurgery implant; or nerve growth factors incorporated into the innercollagenous layer when the prosthesis is used as a conduit for nerveregeneration. In addition to or in substitution for drugs, matrixcomponents such as proteoglycans or glycoproteins or glycosaminoglycansmay be included within the construct.

[0056] The tissue repair fabric can be laser drilled to create micronsized pores, through the completed prosthesis for aid in cell ingrowthusing an excimer laser (e.g. at KrF or ArF wavelengths). The pore sizecan vary from 10 to 500 microns, but is preferably from about 15 to 50microns and spacing can vary, but about 500 microns on center ispreferred. The tissue repair fabric can be laser drilled at any timeduring the process to make the prosthesis, but is preferably done beforedecontamination or sterilization.

[0057] Voids or spaces can also be formed by the method of phaseinversion. At the time of layering the ICL, between layers isdistributed crystalline particles that are insoluble in the liquid heatsource for bonding but should be soluble in the quench bath or thecrosslinking solution. If laser or dry heat is used to bond the layersthen any soluble crystalline solid may be used as long as it is solublein the quench bath or the crosslinking solution. When the crystallinesolid is solubilized and has diffused out, there remains a spacein-which the solid had occupied. The size of the particles may vary from10 to 100 microns, but is preferably from about 15 to 50 microns andspacing can vary between particles when distributed between the layers.The number and size of the voids formed will also affect the physicalproperties (i.e., compliance, kink resistance, suture retention,pliability).

[0058] The following examples are provided to better elucidate thepractice of the present invention and should not be interpreted in anyway to limit the scope of the present invention. Those skilled in theart will recognize that various modifications, can be made to themethods described herein while not departing from the spirit and scopeof the present invention.

EXAMPLES Example 1 Harvesting and Processing of the Intestinal CollagenLayer From Porcine Intestine

[0059] The small intestine of a pig was harvested and mechanicallystripped, using a Bitterling gut cleaning machine (Nottingham, UK) whichforcibly removes the fat, muscle and mucosal layers from the tunicasubmucosa using a combination of mechanical action and washing using hotwater. The mechanical action can be described as a series of rollersthat compress and strip away the successive layers from the tunicasubmucosa when the intact intestine is run between them. The tunicasubmucosa of the small intestine is harder and stiffer than thesurrounding tissue, and the rollers squeeze the softer components fromthe submucosa. The result of the machine cleaning was such that thesubmucosal layer of the intestine solely remained. Finally, thesubmucosa was decontaminated with 0.3% peracetic acid for 18 hours at 4°C. and then washed in phosphate buffered saline. The product thatremained was an intestinal collagen layer (ICL).

Example 2 Various Welding Temperatures and EDC Concentrations of ICL

[0060] The effects of welding temperature (followed by quenching), weldtime, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC)concentration, acetone concentration and crosslinking time, afterwelding on weld strength were examined for the ICL two layered tubeapplication. ICL was porcine derived as described in the Example 1.Strength qualities were measured using a suture retention test and aultimate tensile strength (UTS) test.

[0061] ICL was inverted and stretched over a pair of mandrels which wereinserted into an ICL mounting frame. Mandrels were of stainless steeltubing with an external diameter of 4.75 mm. The ICL and mandrels werethen placed in a dehydration chamber set at 20% relative humidity at 4°C. for about 60 minutes. After dehydration, the ICL was removed from thechamber and the mandrels. The lymphatic tag areas were removed and theICL was manually wrapped around the mandrel twice to form an ‘unwelded’bilayer construct. The wrapped ICL was returned to the dehydrationchamber and allowed to dry for another 90 minutes still at 20% relativehumidity to about 50% moisture +/−10%. To determine the amount ofmoisture present in a sample construct, a CEM™ oven was used.

[0062] A THERMOCENTER™ oven was set for the designated temperaturetreatment for the constructs to be welded. Temperatures tested forwelding ranged from 55° to 70° C. Once the constructs were placed in theoven, the oven was allowed to equilibrate before timing began. Theconstructs were allowed to remain in the chamber for the time requiredfor that condition. Welding times ranged from 7 to 30 minutes. As soonas the time was completed the constructs were removed from the chamberand placed in a 4° C. water bath for about 2 to 5 minutes. The weldedconstructs were then returned to the dehydration chamber for about 30minutes until dehydrated to about 20% +/−10%.

[0063] After dehydration, constructs were inserted into a vesselcontaining EDC in either deionized water or deionized water and acetoneat the concentrations appropriate for the conditions tested. EDCconcentrations tested were 50, 100, and 200 mM. Acetone concentrationstested were 0, 50, and 90% in water. The time duration for crosslinkingwas determined by the conditions tested. Crosslinking times were 6, 12,and 24 hours. After crosslinking, the construct was removed from thesolution and rinsed with physiological pH phosphate buffered saline(PBS) three times at room temperature. The welded and crosslinkedconstruct was then removed from the mandrel and stored in PBS untiltesting. In addition to the thirty constructs that were prepared, twoother bilayer constructs were prepared by welding at 62° C. for 15minutes and crosslinked in 100 mM EDC in 100% H₂O for 18 hours.

[0064] The suture retention test was used to determine the ability of aconstruct to hold a suture. A piece of construct was secured in aCHATTILION™ force measurement device and 1-2 mm bite was taken with aSURGLENE™ 6-0 suture, pulled through one wall of the construct andsecured. The device then pulls at the suture to determine the forcerequired to tear the construct material. The average suture breaksbetween 400-500 g of force; the surgeons pull tends to be 150 g offorce.

[0065] The weld/material strength test was performed to determine theUTS of a construct. Sample rings of 5 mm lengths were excised from eachtube and each was tested for their ultimate tensile strength (UTS) testusing a mechanical testing system MTS™. Three sample rings were excisedfrom each tube for three test pulls done for each construct for a totalof 90 pulls. A ring was placed in the grips of the MTS™ and is pulled ata rate of 0.02 kg_(force)/sec until the weld slips or breaks, or untilthe material (rather than the weld) breaks.

Example 3 Various Welding Temperatures of ICL

[0066] The effect of welding temperature and quenching after welding onweld strength were examined for the ICL two layered tube application.

[0067] An ICL sample of 10 feet long was cut along its length andprepared as in the procedure outlined in Example 2. Six 6 mm diametertubes ranging between 15-20 cm in length were prepared for eachtemperature condition.

[0068] Tubes were subjected to a temperature condition while wet for 3.5hours. Temperatures conditions were: Room temperature (20° C.), 55° C.,62° C. and 62° C. then immediately quenched in 4° C. bath for oneminute. All tubes were then crosslinked in EDC. Six tubes were placedtogether in 300 mL 100 mM EDC overnight at room temperature. Tubes werethen rinsed with physiological strength phosphate buffered saline aftercrosslinking.

[0069] Sample rings of 5 mm lengths were excised from each tube and eachwas tested for ultimate tensile strength (UTS) test using a MTS™. Fivesample rings were taken from each tube for 5 test pulls on each of 6tubes per condition for a total of 30 pulls.

[0070] Weld strength was less consistent for tubes bonded by dehydrationat room temperature as compared to the other temperature treatments whentested using the UTS test. One of the six tubes welded at roomtemperature had UTS measurements comparable to those of the othertreatments. For the tubes welded at other temperatures either with orwithout quenching, there were no differences in weld strength. After UTStesting, it was determined that the breaking of the material was not aseparation of the weld but a material failure in all instances.

Example 4 The Antigenicity of Crosslinked Intestinal Collagen Layer

[0071] Fresh samples of porcine submucosal intestinal layer wereobtained after the cleaning step as described in example 1. Samples werethen left untreated and stored in water, soaked in physiologicalstrength phosphate buffered saline, treated with 0.1% peracetic acid, orwere treated with 0.1% peracetic acid and then crosslinked with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).Samples were then extracted with a solution of 0.5 M NaCl/0.1 M tartaricacid for about 18 hours.

[0072] Two 12% Tris-glycine sodium dodecylsulfate-polyacrylamide gels(Novex Precast Gels cat# EC6009) were run and then transferred afterabout 18 hours to 0.45 μ nitrocellulose paper. Tartaric acid extracts ofeither untreated or treated ICL were run against a control standard lanecontaining: 10 μl Kaleidoscope Prestained Standards (Bio-Rad cat#161-0324): 2 μl biotinylated SDS-PAGE low range molecular weightstandards (Bio-Rad cat# 161-0306): 6 μl loading buffer; 10 μl of controlstandard were loaded to each lane. The gel was blotted for about 2 hourswith 1% dry non-fat milk (Carnation) in phosphate buffered saline. Thegel was then washed three times with borate buffered saline/Tween with200 μl of wash per lane. Primary antibody in 200 μl of Rb serum andborate buffered saline (100 mM boric acid: 25 mM sodium borate: 150 mMNaCl)/Tween was added to each lane at various titer range (1:40, 1:160,1:640 and 1:2560). The gel was then incubated at room temperature forone hour on a rocker platform (Bellco Biotechnology) with the speed setat 10. The gel was then washed again three times with borate bufferedsaline/Tween. Secondary antibody, goat anti-rabbit Ig-AP (SouthernBiotechnology Associates Inc. cat# 4010-04) at a 1:1000 dilution wasadded to lanes at 200 μl per lane and the gel was incubated for one hourat room temperature on a rocker platform. The nitrocellulose membranewas then immersed in AP color development solution while incubated atroom temperature on a rocker platform until color development wascomplete. Development was stopped by washing the membrane in deionizedwater for ten minutes on a rocker platform while changing the water onceduring the ten minutes. The membrane was then air dried.

[0073] The results obtained from analysis of the gel suggest that theantigenicity of the porcine derived ICL treated with peracetic acid andEDC has greatly reduced antigenicity as compared to the othertreatments.

Example 5 Six Layered Tissue Repair Fabric as an Abdominal Wall Patch

[0074] Six layers of porcine intestinal collagen were superimposed ontoone another on a glass plate. A second plate of glass was then placed ontop of the intestinal collagen layers and clamped tightly to the firstplate. The apparatus was placed into a conventional type oven at 62° C.for one hour. Immediately following heating, the apparatus was placedinto a 4° C. water bath for ten minutes. The apparatus was disassembled,the intestinal collagen layers removed, and treated with 100 mM1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in50% acetone for four hours at 25° C. The material was bagged andsterlized by gamma irradiation (2.5 Mrad).

[0075] The tissue repair fabric was sutured in a 3 cm×5 cm defect in themidline of New Zealand White rabbits (4 kg) using a continuous 2-0prolene suture. Animals were sacrificed at four weeks, ten weeks, and 16weeks, and examined grossly, mechanically, and histologically. Grossexamination showed minimal inflammation and swelling. The graft wascovered with a glistening tissue layer which appeared to be continuouswith the parietal peritoneum. Small blood vessels could be seenproceeding circumferentially from the periphery to the center of thepatch. Mechanically the graft was stable with no reherniation observed.Histological examination revealed relatively few inflammatory cells andthose that were observed were primarily near the margin of the graft(due to the presence of prolene suture material). The peritoneal surfacewas smooth and covered entirely by mesothelium.

Example 6 Two Layered Tissue Repair Fabric as a Pericardial Repair Patch

[0076] Two layers of porcine intestinal collagen were superimposed ontoone another on a glass plate. A second plate of glass was then placed ontop of the intestinal collagen layers and clamped tightly to the firstplate. The apparatus was placed into a conventional type oven at 62° C.for one hour. Immediately following heating, the apparatus was placedinto a 4° C. water bath for ten minutes. The apparatus was disassembled,the intestinal collagen layers removed, and treated with 10 mM1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in50% acetone for four hours at 25° C. The material was bagged andsterilized by gamma irradiation (2.5 Mrad).

[0077] A 3×3 cm portion of New Zealand white rabbit pericardium wasexcised and replaced with a same size piece of tissue repair fabric(anastomosed with interrupted sutures of 7-0 prolene). Animals weresacrificed at four weeks and at 180 days, examined grossly,mechanically, and histologically. Gross examination showed minimalinflammation and swelling. Small blood vessels could be seen proceedingcircumferentially from the periphery to the center to the graft.Mechanically, the graft was stable without adhesion to either thesternum or pericardial tissue. Histological examination revealedrelatively few inflammatory cells and those that were observed wereprimarily near the margin of the graft (due to the presence of prolenesuture material).

Example 7 Hernia Repair Device

[0078] A prototype device for hernia repair was developed using ICL tohave a hollow inner region. The device, when completed, had a roundconformation bonded at the periphery and a swollen inner region renderedswollen by the inclusion of physiological strength phosphate bufferedsaline. The inner region can optionally accommodate a wire coil foradded rigidity or other substance for structural support or delivery ofsubstance.

[0079] To assemble ICL multilayer sheets, 15 cm lengths of ICL weretrimrned of lymphatic tags and cut down the side with the tags to form asheet. Sheets were patted dry with Texwipes. On a clean glass plate(6″×8″), sheets were layered mucosal side down. In this case, twotwo-layer and two four-layer patches were constructed by layering eithertwo or four layers of ICL on the glass plates. A second glass plate(6″×8″) was placed on top of the last ICL layer and the plates wereclamped together and then placed in a hydrated oven at 62° C. for onehour. Constructs were then quenched in deionized water at 4° C. forabout ten minutes. The glass plates were then removed from the bath anda plate removed from each patch. The now bonded ICL layers were thensmoothed out to remove any creases or bubbles. The glass plate wasreplaced upon the ICL layers and returned to the hydrated oven for 30-60minutes until dry. Patches were removed from the oven and partiallyrehydrated by spraying with physiological strength phosphate bufferedsaline.

[0080] For the construction of a bi-layer construct, one bi-layer patchwas removed from the glass plates and placed upon the other bi-layerpatch still on the other glass plate. An annular plate (d_(out)=8.75 cm;d_(in)=6 cm) was placed upon the second patch. About 10 cc ofphysiological strength phosphate buffered saline was then injectedthrough a 25 gauge needle between the two bilayer patches. A secondglass plate was then placed on top of the annular plate and were thenclamped together. For the construction of a four-layer construct, thesame steps were followed except that two four-layer patches were usedrather than two bi-layer patches. The constructs were placed in ahydrated oven at 62° C. for one hour. Constructs were then quenched indeionized water at 4° C. for about fifteen minutes. Constructs were thencrosslinked in 200 mL 100 mM EDC in 50% acetone for about 18 hours andthen rinsed with deionized water. The constructs were then trimmed toshape with a razor blade to the size of the outer edge of the annularplate.

Example 8 Intervertebral Disc Replacement ICL, dense fibrillar collagenand hyaluronic acid were configured to closely approximate the anatomicstructure and composition of an intervertebral disc.

[0081] Dense fibrillar collagen diskettes containing hyaluronic acidwere prepared. 9 mg hyaluronic acid sodium salt derived from bovinetrachea (Sigma) was dissolved in 3 mL 0.5 N acetic acid. 15 mL of 5mg/mL collagen (Antek) was added and mixed. The mixture was centrifugedto remove air bubbles. To ,three transwells (Costar) in a six well plate(Costar) was added 5 mL of the solution. To the area outside thetranswell was added N600 PEG to cover the bottom of the membranes. Theplate was maintained at 4° C. on an orbital shaker table at low speedfor about 22 hours with one exchange of PEG solution after 5.5 hours.PEG solution was removed and the transwells dehydrated at 4° C./20% Rhovernight.

[0082] To assemble ICL multilayer sheets, 15 cm lengths of ICL weretrimmed of lymph tags and cut down the side with the tags to form asheet. Sheets were patted dry with Texwipes. On a clean glass plate,sheets were layered mucosal side down to five layers thick and a secondglass plate was laid on top of the fifth layer. Five five-layer patcheswere constructed. The plates with the ICL between were clamped togetherand placed in a hydrated oven at 62° C. for one hour. Constructs werethen quenched in RODI water at 4° C. for about ten minutes then wereremoved form the quench bath and stored at 4° C. until assembly of thedisc.

[0083] To another glass plate, one large patch was laid. A slightlysmaller patch was laid upon the first patch aligned to one edge of thelarger patch. One patch was cut in half and a hole was cut in the centerof each approximating the size of the DFC diskettes. With the centerholes aligned, the two halves were laid upon the second patch aligned tothe same edge. Three rehydrated DFC/HA diskettes were placed within thecenter hole. Another slightly smaller patch was laid upon the two halvescontaining the DFC diskettes and a larger patch laid upon the smallerpatch, both aligned to the same edge. A second glass plate was placed ontop of the construct. The resultant shape was that of a wedge with thethicker side being the one with the aligned edges tapering to theopposite side. The thus formed device was placed in a hydrated oven at62° C. for one hour and then quenched in RODI water at 4° C. for aboutten minutes. The device was then crosslinked in 100 mM EDC (Sigma) in90% acetone (Baxter) for about five hours and then rinsed with threeexchanges of phosphate buffered saline. The edges of the device werethen trimmed with a razor blade.

Example 9 The Formation of Vascular Graft Construct

[0084] The proximal jejunum of a pig was harvested and processed with aGut Cleaning Machine (Bitterling, Nottingham, UK) and thendecontaminated with peracetic acid solution as described in example 1.The peracetic acid treated ICL (PA-ICL) was cut open longitudinally andlymphatic tag areas were removed to form a sheet of ICL. The ICL sheetswere wrapped around a 6.0 mm diameter stainless steel mandrels to formbilayer constructs. The constructs (on mandrels) were then placed in anequilibrated THERMOCENTER™ oven chamber set at 62° C. for about 1 hour.The constructs were removed from the chamber and placed in a 4° C. waterbath for about 2 to 5 minutes. The constructs were chemicallycrosslinked in 50 mL of 100 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in 50/50 water/acetone solution for 18 hours to formperacetic acid treated, EDC crosslinked (PA/EDC-ICL) vascular graftconstructs. The constructs were removed from the mandrels and rinsedwith water to remove residual EDC solution.

[0085] After removal from the mandrels, a layer (approximately 200 μmthick) of type I collagen extracted from bovine tendon, was deposited onthe luminal surface of the constructs according to the method describedin U.S. Pat. No. 5,256,418, incorporated herein. Polycarbonate barbs(luer lock fittings that are conically shaped on one end) were sealablyfixed at either end of the constructs and the constructs were placedhorizontally in a deposition fixture. A 50 mL reservoir of 2.5 mg/mLacid-extracted collagen, prepared by the method described in U.S. Pat.No. 5,106,949, incorporated herein, was attached via the barbs. Thecollagen was allowed to fill the lumen of the ICL tube and was thenplaced into a stirring bath of 20% MW 8000 polyethylene glycol (SigmaChemicals Co.) for 16 hours at 4° C. The apparatus was then dismantledand a 4 mm diameter glass rod was placed into the collagen-filled ICLtube to fix the luminal diameter. The prosthesis was then allowed todry.

[0086] The luminal DFC layer was coated with benzalkonium chlorideheparin (HBAC) by dipping the grafts three times into an 800 U/mLsolution of HBAC and allowed to dry. Finally, the graft received a finalchemical sterilization treatment in 0.1% v/v peracetic acid. The graftwas stored in a dry state until the implant procedure.

Example 10 Implant Studies on Animal Models

[0087] Twenty-five mongrel dogs weighing 15-25 kg were fasted overnightand then anesthetized with intravenous thiopental (30 mg/kg), entubated,and maintained with halothane and nitrous oxide. Cefazolin (1000 mg) wasadministered intravenously preoperatively as well as postoperatively.Each dog received either an aortic bypass grafts or a femoralinterposition graft. For the aortic bypass grafts, a midline abdominalincision was made and the aorta exposed from the renal arteries to thebifurcation, followed by the administration of intravenous heparin (100U/kg). The grafts (6 mm×8 cm) were, placed between the distal infrarenalaorta (end-to-side anastomosis) and the aorta just proximal to thebifurcations (end-to-side anastomosis). The aorta was ligated distal tothe proximal anastomosis. The incisions were closed and the dogsmaintained on aspirin for 30 days post surgery. For the femoralinterposition grafts, the animals were opened bilaterally, the femoralarteries exposed, and a 5 cm length excised. The grafts (4 mm×5 cm) wereanastomosed in end-to-end fashion to the femoral artery. On thecontralateral side, a control graft was placed. The incisions wereclosed and the animals were maintained on aspirin for 30 days postsurgery. Post operative follow-up ranged from 30 days to 360 days.Pre-implant, and four and eight weeks post-implant bloods werecollected. Animals were sacrificed at various time points (30 days, 60days, 90 days, 180 days, and 360 days).

[0088] New Zealand White rabbits weighing 3.5-4.5 kg were fastedovernight, and then anesthetized with acepromazine (20 mg) and ketamine(40 mg), entubated, and maintained with ketamine (50 mg/mL), injectedintravenously as needed. Penicillin (60,000 U) was administeredintramuscularly preoperatively. A midline abdominal incision was madeand the aorta exposed from the renal arteries to the bifurcation,followed by the administration of intravenous heparin (100 U/kg). A 3 cmlength of aorta was excised, and the grafts (2.5 mm×3 cm) wereanastomosed in end-to-end fashion to the aorta. The incisions wereclosed and the animals were maintained with no anticoagulant therapypost surgery. Post operative follow-up ranged from 30 days to 360 days.Animals were sacrificed at various time points (30 days, 60 days, 90days, 180 days, and 360 days).

[0089] The implants along with adjacent vascular tissues obtained fromsacrificed animals were fixed for transmission electron microscopy (TEM)analysis for 4 hr in a solution of 2.0% paraformaldehyde, 2.5%glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4. Samples were thenpost-fixed in 1.0% OsO4 (in 0.1M sodium cacodylate) and stained en blocwith 2.0% uranyl acetate (aqueous). After secondary fixation allspecimens were dehydrated in a graded ethanol series and propylene oxideand embedded in Epox 812 (Ernest F. Fullam, Rochester, N.Y., USA).Ultrathin (˜700 nm) sections were stained with uranyl acetate and leadcitrate. Sections were examined on a JEOL Instruments JEM100S at 80 kV.For scanning electron microscopy (SEM), samples were fixed for 18 hr inhalf strength Karnovsky's solution and rinsed five times in Sorensen'sphosphate buffer prior to post fixation in 1.0% OsO4 for 1 hr. Sampleswere then rinsed twice in Sorensen's phosphate buffer and three times indouble distilled water. Dehydration was accomplished through an ethanolseries (50%, 70%, 90%, and 100%), followed by critical point drying.Samples were mounted and coated with 60/40 gold/palladium.

[0090] ICL graft explants from dogs and rabbits were examinedhistologically to evaluate host cell ingrowth. Masson's trichromestaining of a 60 day explant showed significant host infiltrate. Thedarker blue staining showed collagen of the ICL while matrix surroundingthe myofibroblasts, stained lighter blue, showed an abundance of hostcollagen. High power magnification of the section showed numerous cellsintermingled within the ICL. The inflammatory response seen at 30 dayshad been resolved and the cellular response was predominantlymyofibroblastic. The surface of the remodeled graft was lined byendothelial cells as demonstrated by SEM and Factor VIII staining. By360 days, a mature ‘neo-artery’ had been formed. The neo-adventitia wascomposed of host collagen bundles populated by fibroblast-like cells.The cells and matrices of the remodeled construct appeared quite matureand tissue-like.

Example 11 Generation of Anti-ICL Antibodies

[0091] Fresh samples of porcine submucosal intestinal layer wereobtained after the cleaning step as described in example 1 but were notperacetic acid treated. Samples were then left untreated (NC-ICL),treated with 0.1% peracetic acid (PA-ICL), or treated with 0.1%peracetic acid and then crosslinked with1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride(PA/EDC-ICL).

[0092] New Zealand White rabbits were immunized with 0.5 mg of any oneof the three types of ICL samples (NC-ICL, PA-ICL, or PA/EDC-ICL) togenerate anti-serum. Initially, rabbits were injected subcutaneouslywith 0.5 mL of homogenized untreated ICL ini Freund's complete adjuvant(1:1, 1 mg/mL). Sham rabbits received 0.5 mL of phosphate bufferedsaline in Freund's complete adjuvant. Rabbits were boosted every 3 to 4months with 0.5 mL of the appropriate form of ICL in Freund's incompleteadjuvant (0.25 mg/mL). Sera were collected 10-14 days after each boost.

Example 12 Generation of ICL Extracts and Characterization ofPotentially Antigenic Proteins Associated With Native Collagen

[0093] Proteins were extracted from NC-ICL, PA-ICL, or PA/EDC-ICL usingtartaric acid (Bellon, G., et al (1988) Anal. Biochem . 175: 263-273) orTRITON X-100 (Rohm and Haas). Pulverized NC-ICL, PA-ICL, or PA/EDC-ICL(10% w/v) were mixed with either tartaric acid (0.1 M tartaric acid, 0.5M NaCl) or TRITON X-100 (Rohm and Haas) extraction buffer (TEB; 1%TRITON X-100 in 20 mM Tris HCl (pH 7.2), 2 mM EGTA, 2 mM EDTA, 1 mMphenylmethylsulfonyl fluoride, and 25 mg/mL each of aprotinin,leupeptin, and pepstatin (Sigma, St. Louis, Mo.)). The mixtures wereincubated overnight at 4° C. The extracts were gauze filtered to removedebris, dialyzed against PBS and concentrated using Centriprep-30(Amicon, Danvers, Mass.). Extracts were stored at −80° C. until used.

[0094] Tartaric acid and TEB extracts of were separated on 10%polyacrylamide gels by SDS-PAGE according to Laemmli (Laemmli, U.K.(1970) Nature 227: 680 -685). Gels were either silver stained (Bio-Rad,Hercules, Calif.) or transferred to nitrocellulose membranes (Amersham,Arlington Heights, Ill.). Multiple protein bands were visualized in theNC-ICL extracts by silver staining. In contrast, only two bands werevisible in the PA-ICL extracts and no protein bands were seen in thelanes containing PA/EDC-ICL. These results suggest that peracetic acidand EDC treatment, in combination, leads to a decrease in theextractable non-collagenous proteins in ICL. ihmunoblot transfer wasdone overnight using a Trans-Blot Cell (Bio-Rad) in Tris-Glycine 20%methanol transfer buffer. Nitrocellulose membranes containing ICLtransferred proteins were blocked with Blotto buffer (1% non-fat drymilk in borate buffered saline with 0.1% Tween-20 (BBS/Tween)) for onehour at room temperature. The nitrocellulose membranes were transferredto a multiscreen apparatus containing 12 individual lanes. The membraneswere washed three times with BBS/Tween. Positive control or test sera(100 μL/lane) were added to the membrane and rocked at room temperaturefor 1 hour. Each lane was washed three times with BBS/Tween. Secondaryantibodies: ALPH-labeled goat anti-rabbit Ig or ALPH-labeled goatanti-dog Ig (Southern Biotechnology) were added to the appropriate lanes(100 μL/lane) and streptavidin-AP (100 μL) was added to one of the lanescontaining the Kaleidoscope molecular weight standards (Bio-Rad). Analkaline phosphatase conjugate substrate kit (Bio-Rad) was used tovisualize the immunoblots.

[0095] Rabbit anti-NC-ICL serum, generated by repeated immunization withNC-ICL, was used to detect potentially immunoreactive proteins. Serafrom immunized rabbits recognized antigens with molecular weights in therange of <30, 40-70, and >100 kDa in the tartaric acid extract. Thesesame sera were tested on immunoblots of TEB extracts from NC-ICL.Immunoreactive proteins were detected with molecular weights rangessimilar to those detected in the tartaric acid extract, with additionalreactivity detected in the 70-100 kDa range. The results indicated thatNC-ICL contains multiple proteins which are immunoreactive and theseproteins can be extracted by tartaric acid or TEB. The greater number ofimmunoreactive proteins present in the TEB extract correlated with theincrease in proteins extracted using TEB as compared to tartaric acid.

Example 13 Effect of PA or EDC Treatment of ICL on the Antigenicity ofType I Collagen in ICL

[0096] Sera from rabbits immunized with NC-ICL, PA-ICL, or PA/EDC-ICL(sera prepared as described in example 11) or acid extracted type Icollagen (Organogenesis, Canton, Mass.) were tested for type I collagenspecific antibodies by ELISA. ELISA plates (Immulon II, NUNC,Bridgeport, N.J.) were coated with 200 mLswell of 1 mg/mL acid extractedtype I collagen in 0.05 M carbonate buffer (pH 9.6) overnight at 4° C.Plates were washed twice with PBS/Tween-20 (0.1%). Serum samples fromanimals or rabbit anti-collagen type I antibody (Southern Biotechnology,Birmingham, Ala.) were added to wells (100 mL/well) and incubated for 1hr at room temperature. Plates were washed three times with PBS/Tween.Secondary antibodies: ALPH-labeled goat anti-rabbit Ig or ALPH-labeledgoat anti-dog Ig (Southern Biotechnology) were added to the appropriatewells and incubated at room temperature for 1 hour. Plates were washedthree times with PBS/Tween. P-nitrophenylphosphate (PNPP) substrate (1mg/mL) was added to each well (100 mL/well). Absorbance was read at 405nm on a SpectraMax microplate reader (Molecular Devices, Sunnydale,Calif.).

[0097] Anti-collagen type I antibodies could not be detected in serafrom rabbits immunized with any form of ICL, even at a 1:40 serumdilution. In contrast, rabbits immunized with purified type I collagenhad antibody titer of 1:2560. These data suggest that crosslinking isnot necessary to reduce the antigenicity to collagen type I, sincerabbits immunized with NC-ICL did not generate anti-collagen type Iantibodies. These data thus suggest that the immunodominant proteins inNC-ICL are non-collagenous proteins. Also, the effect of PA and EDC onreducing the antigenicity of ICL is directed toward the non-collagenousproteins.

Example 14 Effects of Disinfecting and Crosslinking on Antigenicity ofICL

[0098] The effect of PA and EDC treatment on the antigenicity of ICL wasdetermined by using anti-NC-ICL antiserum to probe for immunoreactiveproteins present in tartaric acid or TEB extracts of PA or PA/EDCtreated ICL.

[0099] Tartaric acid extracts of PA-ICL and TEB extracts of PA/EDC-ICLwere separated on 10% SDS-PAGE gels and transferred to nitrocellulosemembranes for immunoblot analysis, as described in Example 12. NC-ICLspecific antisera were used to probe for immunoreactive proteins in eachextract. Even when immunoblots of PA-ICL. and PA/EDC-ICL wereoverexposed, no reactivity could be detected in lanes containinganti-NC-ICL antibodies thus suggesting that the immunoreactive proteinsdetected in the NC-ICL are either missing or their epitopes have beenmodified such that they are no longer recognized by anti-NC-ICLanti-serum. To address this latter issue, serum from rabbits immunizedwith either PA-ICL or PA/EDC-ICL was also tested. No antibody bindingwas detected in any of the lanes above background. These data indicatethat even when rabbits were immunized with modified ICL they did notgenerate antibodies which could recognize modified ICL extractedproteins. These results suggest that the proteins removed or modifiedduring the process of disinfecting and crosslinking are the sameproteins responsible for the antigenicity of NC-ICL.

[0100] Antibody response of PA-ICL or PA/EDC-ICL immunized rabbits wasanalyzed by immunoblotting, as described in Example 12. This approachwas taken to ensure that the lack of reactivity of anti-NC-ICL sera withPA/EDC-ICL was due to the absence of proteins in ICL and not due to aninability to extract proteins which might be accessible to the immunesystem in vivo since crosslinking of collagenous materials with EDCcould reduce the quantity and quality of protein extracted from ICL.Anti-ICL antisera was generated using PA-ICL or PA/EDC-ICL to immunize-rabbits. Sera from these rabbits were tested for antibodies specific forproteins in either tartaric acid or TEB protein extracts of NC-ICL.Anti-PA-ICL recognized the 207, 170, and 38-24 kDa proteins recognizedby anti-NC-ICL, but lost reactivity to the lower molecular weightproteins. No bands were detected by the anti-PA/EDC-ICL serum from 1rabbit. Serum from another anti-PAIEDC-ICL rabbit reacted with the 24-38kDa proteins. These data suggested that both PA-ICL and PA/EDC-ICL areless antigenic than NC-ICL. Either the antigenic epitopes of ICL areremoved during the disinfecting and crosslinking process or they aremodified to reduce their antigenicity. In either case, disinfection andcrosslinking resulted in a material whose antigenicity was significantlyreduced.

Example 15 Determination of Humoral Immune Response in Graft Recipients

[0101] Dogs were tested for a humoral immune response to ICL graftcomponents to determine if ICL must retain its antigenicity to stimulatecell ingrowth into the graft. Pre-implant, and four and eight weekspost-implant blood samples were collected from fifteen dogs thatreceived PAIEDC-ICL vascular grafts. Serum from each blood sample wastested for antibodies to proteins in both the tartaric acid and TEBextracts of NC-ICL. Even at a 1:40 dilution of serum, none of the dogstested had antibodies which reacted with ICL proteins. These same serumsamples were tested for the presence of anti-collagen type I antibodiesby ELISA. All serum samples were negative for antibodies to type Icollagen at a serum dilution of 1:40. Masson's trichrome staining ofexplant paraffin sections from these dogs did shown infiltration of hostcells. These results demonstrate that PA/EDC-ICL does not elicit anantibody response when the host is actively remodeling the material.

[0102] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be obvious to one of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

What is claimed:
 1. A prosthesis comprising two or more superimposed,bonded layers of collagenous tissue which have been crosslinked with acrosslinking agent that permits bioremodeling and sterilized, whereinall of the layers of the prosthesis are completely bioremodelable, andwhich, when implanted into a mammalian patient, undergoes controlledbiodegradation occurring with adequate living cell replacement such thatthe original implanted prosthesis is remodeled by the patient's livingcells.
 2. The prosthesis of claim 1 wherein the shape of said prosthesisis flat, tubular, or complex.
 3. The prosthesis of claim 1 wherein saidcollagen material is sourced from a mammalian source and is intestinalmaterial, fascia lata, dura mater, and pericardium.
 4. The prosthesis ofclaim 3 wherein said collagen material is the tunica submucosa of thesmall intestine.
 5. The prosthesis of claim 1 wherein said collagenlayers are bonded together by heat welding for a time and underconditions sufficient to effect the bonding of the collagenous tissuelayers.
 6. The prosthesis of claim 1 wherein said prosthesis iscrosslinked with the crosslinking agent1-ethyl-3-(3-dimethylaminopropyl) carbodjimide hydrochloride.
 7. Theprosthesis of claim 6 wherein sulfo-N-hydroxysucciniimide is added tothe crosslinking agent.
 8. The prosthesis of claim 6 wherein acetone isadded to the crosslinking agent.
 9. The prosthesis of claim 1 whereinthe prosthesis is sterilized with peracetic acid.
 10. The prosthesis ofclaim 10 wherein said prosthesis is non-antigenic.
 11. The prosthesis ofclaim 1 wherein one or more surfaces of said prosthesis is coated with acollagenous material which acts as a smooth flow surface.
 12. Theprosthesis of claim 1 wherein said prosthesis further contains pores.13. The prosthesis of claim 1 wherein said prosthesis is furthercomposed of chopped collagen fibers.
 14. The prosthesis of claim 1wherein said prosthesis is further composed of collagen threads.
 15. Theprosthesis of claim 14 wherein said collagen threads are arranged toform a felt, a bundle, a weave or a braid.
 16. The prosthesis of any ofclaims 13-15 wherein said collagen fibers or threads are partially orcompletely crosslinked.
 17. The prosthesis of claim 1 wherein saidprosthesis additionally contains an anticoagulant; one or moreantibiotics, or one or more growth factors.
 18. A method of preparing aprosthesis having two or more superimposed, bonded layers of collagenmaterial, comprising: (a) bonding the two or more collagen layerstogether using heat welding by heating said collagenous tissue layersfor a time and under conditions sufficient to effect the bonding of thecollagen layers and to form a prosthesis; (b) cooling said heatedprosthesis; and, (c) crosslinking said prosthesis with a crosslinkingagent that permits bioremodeling, wherein said thus formed prosthesiswhen implanted into a mammalian patient, undergoes controlledbiodegradation occurring with adequate living cell replacement such thatthe original implanted prosthesis is remodeled by the patient's livingcells; wherein the collagenous tissue layers are sterilized withperacetic acid before bonding in step (a) or the prosthesis issterilized after crosslinking in step (c).
 19. The method of claim 18wherein said collagen layers are formed from two or more layers ofcollagenous tissue sourced from a mammalian source and is intestinalmaterial, fascia lata, dura mater, and pericardium.
 20. The method ofclaim 19 wherein said collagen material is the tunica submucosa of thesmall intestine.
 21. The method of claim 18 wherein said heat welding isfrom about 50° C. to about 75° C., more preferably from about 60° to 65°C. and most preferably at about 62° C.
 22. The method of claim 18wherein said cooling is accomplished by quenching.
 23. The method ofclaim 18 wherein said heat welding is accomplished for a time from about7 minutes to about 24 hours, preferably about 1 hour.
 24. The method ofclaim 18 wherein said prosthesis is crosslinked with the crosslinkingagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. 25.The method of claim 18 wherein said prosthesis is non-antigenic.
 26. Amethod of repairing or replacing a damaged tissue comprising implantinga prosthesis in a patient comprising two or more superimposed, bondedlayers of collagenous tissue which have been sterilized with peraceticacid and crosslinked with a crosslinking agent that permitsbioremodeling, wherein all of the layers of the prosthesis arecompletely bioremodelable, and which, when implanted into a mammalianpatient, undergoes controlled biodegradation occurring with adequateliving cell replacement such that the original implanted prosthesis isremodeled by the patient's living cells.
 27. A sterile, non-pyrogenic,and non-antigenic prosthesis formed from mammalian derived collagenoustissue for engraftment to a recipient patient, whereby said engraftedprosthesis does not elicit a humoral immune response to components insaid collagenous tissue and wherein said prosthesis concomitantlyundergoes bioremodeling occurring with adequate living cell replacementsuch that the original implanted prosthesis is remodeled by thepatient's living cells.
 28. The prosthesis if claim 27 wherein saidhumoral immune response to components derived from said collagenoustissue demonstrates no significant increase in antibody titer forantibodies from baseline titer levels when blood serum obtained from arecipient of a prosthesis is tested for antibodies to proteins inextracts of the collagenous tissue..
 29. The prosthesis of claim 28wherein said antibody titer levels is 1:40 or less for a patient or hostpreviously non-sensitized to collagenous tissue proteins.
 30. A methodof preparing a non-antigenic prosthesis prepared from collagenous tissuederived from a mammalian source selected from the group consisting ofintestinal material, fascia lata, dura mater, and pericardium,comprising: (a) disinfecting the collagen material with peracetic acidat a concentration between about 0.01 and 0.3% v/v in water; and, (b)crosslinking said sterilized collagenous tissue with a crosslinkingagent that permits bioremodeling; wherein the prosthesis isformed fromtwo or more superimposed, bonded layers of collagenous tissue, whereinall of the layers of the prosthesis are bioremodelable, and wherein theprosthesis when implanted into a marmnalian patient, undergoescontrolled bioremodeling occurring with adequate living cell replacementsuch that the original implanted prosthesis is remodeled by thepatient's living cells without eliciting a significant humoral immuneresponse.
 31. The method of claim 30 wherein said collagenous tissue isthe tunica submucosa of the small intestine.
 32. The method of claim 30wherein said collagen material is formed from two or more layers ofsuperimposed, bonded layers of collagen material.
 33. The method ofclaim 30 wherein the prosthesis is sterilized with peracetic acid priorto implantation into the mammalian patient.